Genetic Engineering of the Biosynthesis of Glycine Betaine Modulates Phosphate Homeostasis by Regulating Phosphate Acquisition in Tomato

Abstract

Glycine betaine (GB), as a putative compatible substance, protects plants against the damaging effects of abiotic stresses. Phosphorus deficiency is one type of abiotic stress that is detrimental to plant growth. Maintenance of phosphate (Pi) homeostasis is crucial. This study demonstrates GB-regulated phosphate homeostasis in the tomato (Solanum lycopersicum cv. 'Moneymaker') transformed with the choline oxidase gene codA from Arthrobacter globiformis. The codA-transgenic lines displayed more resistance to low-phosphate stress. The data revealed that the wild-type plants were stunted and consistently retained less Pi than transgenic lines, especially when grown under low-phosphate conditions. This difference in Pi retention was attributable to the enhanced Pi uptake ability in the transgenic lines. The transgenic plants translocated more Pi into the plant cell due to the enhanced enzymatic activity of plasma membrane H⁺-ATPase and increased Pi/H⁺ co-transport, which improved Pi uptake. The differential expression of 'PHO regulon' genes further maintained intracellular Pi homeostasis. Furthermore, GB maintained a higher photosynthesis rate, thus increasing the production and translocation of sucrose via phloem loading to enhance plant response to low-phosphate stress. We conclude that GB mediates Pi uptake and translocation by regulating physiological and biochemical processes that promote adaptation to environmental changes in Pi availability. These processes eventually lead to better growth and development of the codA-transgenic lines. This finding will help to further elucidate the signaling mechanism of how GB perceives and transmits low-phosphate signals to alleviate Pi nutritional stress.

Keywords: codA gene; glycinebetaine; low phosphate stress; phosphate acquisition; phosphate homeostasis; tomato.

FIGURE 1

Levels of glycine betaine (GB) in the leaves of wild-type (WT) plants and three codA-transformed tomato lines (L2, L3, and L4). Plants were treated under normal or low-phosphate stress conditions for 15 days. Values represent the means ± SD of four replicates. Asterisks indicate significant differences compared with WT plants (Student’s t-test). FW, fresh weight; ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 2

Growth phenotype of WT and transgenic lines under variable Pi conditions. (A,B) The shoot and root phenotypes of 15-day-old WT tomato plant and three codA-transgenic tomato lines under variable Pi conditions. Seedlings (after germination) were treated with 1.0 mM phosphorus (CK), 0.2 mM phosphorus and 0.02 mM phosphorus (LP) for 15 days under sand-culture system. Then, photos were taken. (C) The whole-plant biomass of WT and transgenic lines under the various Pi concentrations as described in (A,B). 15-day-old tomato seedlings were collected for biomass analysis. Values are means ± SD. n = 12 for each genotype. (D) Anthocyanin content in the leaves of plants treated with 1.0, 0.2, 0.02 mM phosphorus for 15 days; seedlings were subsequently harvested for measuring anthocyanin content. (E) Starch accumulation of the 15-day-old WT and transgenic lines described in (D). Values represent means ± SD of three replicates. Asterisks indicate significant differences compared with WT plants (Student’s t-test). FW, fresh weight; ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 3

Sucrose concentration (A), SS and SPS activity (B,C) and leaf SUC2 gene expression (D) in WT tomato plants and three codA-transgenic tomato lines under variable Pi conditions. Seedlings (after germination) were treated with 1.0 mM phosphorus (CK), 0.2 mM phosphorus and 0.02 mM phosphorus (LP) for 15 days under sand-culture system. The seedlings were subsequently used for experimental analysis. Values represent means ± SD of three replicates. Asterisks indicate significant differences compared with WT plants (Student’s t-test). SS, sucrose synthesis; SPS, sucrose phosphate synthesis; FW, fresh weight; ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 4

Photosynthetic rate (Pn) of leaves of plants grown with normal or low phosphorus. Seedlings (after germination) were treated with 1.0 mM phosphorus (CK), 0.2 mM phosphorus and 0.02 mM phosphorus (LP) for 15 days under sand-culture system. The photosynthetic rate was measured by the CIRAS-3. Values are means ± SD. n = 6 for each genotype. Asterisks indicate significant differences compared with WT plants (Student’s t-test). ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 5

Comparison of total P and Pi content between WT and transgenic plants. Plants were grown under variable Pi conditions for 15 days. (A) Total phosphorus content of WT and transgenic lines under different Pi concentrations. Seedlings (after germination) were treated with 1.0 mM phosphorus (CK), 0.2 mM phosphorus, and 0.02 mM phosphorus (LP) for 15 days under sand-culture system. Seedlings were subsequently harvested for measuring phosphorus content. Values are means ± SD. n = 4 for each genotype. (B–D) The Pi content of the leaves, stem and root under normal Pi and low Pi conditions. Pi content was measured in harvested 15-day-old tomato plants. Values represent the means ± SD of three replicates. Asterisks indicate significant differences compared with WT plants (Student’s t-test). DW, dry weight; FW, fresh weight; ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 6

Comparison of plasma membrane H⁺-ATPase activity derived from tomato roots. Seedlings (after germination) were treated with 1.0 mM phosphorus (CK), 0.2 mM phosphorus, and 0.02 mM phosphorus (LP) for 15 days under a sand-culture system. Values are means ± SD of three replications per experiment, n = 6 for each genotype. Asterisks indicate significant differences compared with WT plants (Student’s t-test). ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 7

H⁺ flux in tomato roots under variable Pi conditions. (A,C,E) Transient net flux of H⁺ in the root elongation zone of WT plants and three codA-transgenic tomato lines under different Pi concentrations. A continuous flux recording over 10 min was conducted in a corresponding measuring solution (pH 6.0). (B,D,F) The mean rate of H⁺ flux within the measuring periods is shown. Values are means ± SD. n = 6 for each genotype. Asterisks indicate significant differences compared with WT plants (Student’s t-test). ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 8

Quantitative analysis of gene expression of PT1 (A), PT2 (B), PHO1 (C), and UBC24 (D) in the root of the WT tomato plant and three codA-transgenic tomato lines under variable Pi conditions. Seedlings (after germination) were treated with 1.0 mM phosphorus (CK), 0.2 mM phosphorus, and 0.02 mM phosphorus (LP) for 15 days under a sand-culture system. Seedlings were subsequently harvested for experimental analysis. Values represent the means ± SD of three biological replicates. Asterisks indicate significant differences compared with WT plants (Student’s t-test). ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 9

A possible model to show the probable mechanism of how GB regulates phosphate acquisition in transgenic tomato plants under low phosphate stress.